PROJECT SUMMARY/ABSTRACT Communication between neurons and their targets depends on proper synaptic growth and activity. The microtubule cytoskeleton plays a central role in synaptic terminal development, and microtubule dysfunction is associated with many neurological disorders. Neurons contain stable and dynamic microtubules, and these two populations must be properly balanced for synapses to grow and form stable connections. In this proposal, we use a synergistic combination of in vivo genetic analyses and cell-free in vitro biophysical approaches to elucidate the mechanisms by which microtubule dynamics and stability are balanced. We leverage a novel α- tubulin mutant that alters the normal microtubule balance and perturbs synaptic growth. This tubulin mutation disrupts a highly conserved, essential α-tubulin site that is acetylated. Post-translational modifications (PTMs), such as acetylation, have the potential to directly and specifically regulate microtubule stability and dynamics to shape synaptic morphogenesis, yet relatively few microtubule PTMs have been studied. Our preliminary data implicate this previously uncharacterized α-tubulin site in regulating the addition of tubulin dimers to growing microtubule ends, which suggests a novel acetylation-based mechanism to control microtubule dynamics. Based on our preliminary findings, we will test the hypothesis that microtubule dynamics and stability are balanced by α-tubulin acetylation and other known regulators to shape synaptic terminal morphogenesis (Aim 1). We will use the Drosophila neuromuscular junction as a model and investigate the effects of manipulating microtubule dynamics and stability on two different motor neuron types, called type Ib and type Is, whose synaptic terminals have distinct morphologies and transmission properties. Our preliminary data indicate that altering the microtubule cytoskeleton has strikingly different effects on the growth of type Ib and Is synaptic terminals. We will test the hypothesis that stable and dynamic microtubules are uniquely balanced in different neuron types to establish distinct neuron-specific synaptic structures and activities (Aim 2). Combined, our studies will reveal novel mechanisms that regulate synaptic microtubule networks and provide fundamental new insight into the central role that microtubules play in creating diverse synaptic morphologies and functions.